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All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and posttranslationally targeted to the organelle. We have used an in vitro assay to reconstitute protein import into pumpkin (Cucurbita pepo) glyoxysomes, a class of peroxisome found in the cotyledons of oilseed plants, to study the mechanisms involved in protein transport across peroxisome membranes. Results indicate that ATP hydrolysis is required for protein import into peroxisomes; nonhydrolyzable analogs of ATP could not substitute for this requirement. Nucleotide competition studies suggest that there may be a nucleotide binding site on a component of the translocation machinery. Peroxisomal protein import also was supported by GTP hydrolysis. Nonhydrolyzable analogs of GTP did not substitute in this process. Experiments to determine the cation specificity of the nucleotide requirement show that the Mg2+ salt was preferred over other divalent and monovalent cations. The role of a putative protonmotive force across the peroxisomal membrane was also examined. Although low concentrations of ionophores had no effect on protein import, relatively high concentrations of all ionophores tested consistently reduced the level of protein import by approximately 50%. This result suggests that a protonmotive force is not absolutely required for peroxisomal protein import.

Peroxisome biogenesis requires that proteins be transported from their site of synthesis in the cytoplasm to their final location in the peroxisome matrix or membrane. Glyoxysomes are a class of peroxisomes found primarily in germinating seedlings and are involved in mobilizing fatty acids via the glyoxylate cycle and the beta-oxidation pathway. We have used an in vitro assay to study the mechanism(s) of import of proteins into glyoxysomes. Results from this assay indicate that the transport process is time- and temperature-dependent and is specific for peroxisomal proteins. Isocitrate lyase, a glyoxysomal protein, and the leaf-type peroxisomal enzyme glycolate oxidase (GLO) were transported into pumpkin (Cucurbita pepo) glyoxysomes with no apparent differences in efficiency of import. Thus, this in vitro assay appears to be physiologically relevant and correlates well with expected in vivo conditions. Protein import was also energy-dependent and saturable. Nonradiolabeled GLO competed with radiolabeled, in vitro-synthesized GLO for components of the import machinery. Finally, pretreatment of the isolated glyoxysomes with protease virtually abolished subsequent import of GLO. Taken together, these results indicate that a proteinaceous receptor is involved in the import of peroxisomal proteins.